Computational Systems Biology Methods and Protocols.7z

designed a new set of universal primers for vertebrate mitogenomes

by referring its most conservative regions and then amplify mito-

genomes, and its PCR product was used to make probe. Second, we

prepared 2 kb length of libraries. Third, the libraries and probe

were mixed to enrich target mtDNA. Then we sheared the enriched

long-range library to approximately 300–700 bp and followed

downstream library construction steps for sequencing. In the

downstream sequence experiment, an Ion Torrent Personal

Genome Machine (PGM) was used to sequence because it is fast

and relatively inexpensive in terms of each run (not price per base).

Each run using 316 chip generated over 800 Mb for 60 samples,

and the data size for each sample is more than 10 Mb in general.

These generated data was sufficient for de novo assembly of a

complete mitochondrial genome. Although we sequenced with

Ion Torrent platform, the protocol could also be applied to

Fig. 1Schematic pipeline for enriching mitochondrial DNA to high-throughput

sequencing. The green line represents using the pair of long-range PCR

(LR-PCR) amplicons to directly construct a library. Compared to standard

library, the long-range library capture hybridization strategy (LR-LCR) has

modification in library construction 1 and 2. LR-LCR requires a long

fragment during shearing in library construction 1 for capturing high variable

loci and additional library construction 2 for PGM sequencing. In standard

hybridization, there is no construction library 2 and the enriched fragments

directly sequenced

Capture Hybridization of Long-Range DNA Fragments for High-Throughput Sequencing 33